Similarities and differences between the subgenomic and minus-strand promoters of an RNA plant virus

Promoter regions required for minus-strand and subgenomic RNA synthesis have been mapped for several plus-strand RNA viruses. In general, the two types of promoters do not share structural features even though they are recognized by the same viral polymerase. The minus-strand promoter of Alfalfa mosaic virus (AMV), a plant virus of the family Bromoviridae, consists of a triloop hairpin (hpE) which is attached to a 3' tRNA-like structure (TLS). In contrast, the AMV subgenomic promoter consists of a single triloop hairpin that bears no sequence homology with hpE. Here we show that hpE, when detached from its TLS, can function as a subgenomic promoter in vitro and can replace the authentic subgenomic promoter in the live virus. Thus, the AMV subgenomic and minus-strand promoters are basically the same, but the minus-strand promoter is linked to a 3' TLS to force the polymerase to initiate at the very 3'end.     

A conserved hairpin structure in Alfamovirus and Bromovirus subgenomic promoters is required for efficient RNA synthesis in vitro

The coat protein gene in RNA 3 of alfalfa mosaic virus (AMV; genus Alfamovirus, family Bromoviridae) is translated from the subgenomic RNA 4. Analysis of the subgenomic promoter (sgp) in minus-strand RNA 3 showed that a sequence of 37 nt upstream of the RNA 4 start site (nt +1) was sufficient for full sgp activity in an in vitro assay with the purified viral RNA-dependent RNA-polymerase (RdRp). The sequence of nt -6 to -29 could be folded into a potential hairpin structure with a loop represented by nt -16, -17, and -18, and a bulge involving nt -23. By introducing mutations that disrupted base pairing and compensatory mutations that restored base pairing, it was shown that base pairing in the top half of the putative stem (between the loop and bulge) was essential for sgp activity, whereas base pairing in the bottom half of the stem was less stringently required. Deletion of the bulged residue A-23 or mutation of this residue into a C strongly reduced sgp activity, but mutation of A-23 into U or G had little effect on sgp activity. Mutation of loop residues A-16 and A-17 affected sgp activity, whereas mutation of U-18 did not. Using RNA templates corresponding to the sgp of brome mosaic virus (BMV; genus Bromovirus, family Bromoviridae) and purified BMV RdRp, evidence was obtained indicating that also in BMV RNA a triloop hairpin structure is required for sgp activity.     

RNA motifs that determine specificity between a viral replicase and its promoter

The 3' end of brome mosaic virus RNA contains a tRNA-like sequence that directs its RNA synthesis. A stem loop structure in this sequence, stem loop C (SLC), was investigated using NMR, and correlated with its ability to direct RNA synthesis by its replicase. SLC consists of two discrete domains, a flexible stem with an internal loop and a rigid stem containing a 5'-AUA-3' triloop. Efficient RNA synthesis requires the sequence on only one side of the flexible stem and a specific compact conformation of the triloop. A high resolution structure of the triloop places the 5' adenine out in solution, and the 3' adenine within the triloop, held tightly through stacking and unusual hydrogen bonds. This high resolution structure of an RNA promoter from a (+)-strand RNA virus provides new insights into how the RNA-dependent RNA polymerase binds to the RNA to initiate synthesis.     

Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.     

Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA.

Expression of brome mosaic virus (BMV) coat protein and internal genes of many other positive-strand RNA viruses requires initiation of subgenomic mRNA synthesis from specific internal sites on minus-strand genomic RNA templates. Biologically active viral cDNA clones were used to investigate the sequences controlling production of BMV subgenomic RNA in vivo. Suitable duplications directed production of specifically initiated, capped subgenomic RNAs from new sites in the BMV genome. Previously implicated promoter sequences extending 20 bases upstream (-20) and 16 bases downstream (+16) of the subgenomic RNA initiation site directed only low-level synthesis. Subgenomic RNA production at normal levels required sequences extending to at least -74 but not beyond -95. Loss of an (rA)18 tract immediately upstream of the -20 to +16 "core promoter" particularly inhibited subgenomic RNA synthesis. The -38 to -95 region required for normal initiation levels contains repeats of sequence elements in the core promoter, and duplications creating additional upstream copies of these repeats stimulated subgenomic RNA synthesis above wild-type levels. At least four different subgenomic RNAs can be produced from a single BMV RNA3 derivative. For all derivatives producing more than one subgenomic RNA, a gradient of accumulation progressively favoring smaller subgenomic RNAs was seen.     

A minimal RNA promoter for minus-strand RNA synthesis by the brome mosaic virus polymerase complex

The approximately 150 nt tRNA-like structure present at the 3' end of each of the brome mosaic virus (BMV) genomic RNAs is sufficient to direct minus-strand RNA synthesis. RNAs containing mutations in the tRNA-like structure that decrease minus-strand synthesis were tested for their ability to interact with RdRp (RNA-dependent RNA polymerase) using a template competition assay. Mutations that are predicted to disrupt the pseudoknot and stem B1 do not affect the ability of the tRNA-like structure to interact with RdRp. Similarly, the +1 and +2 nucleotides are not required for stable template-RdRp interaction. Mutations in the bulge and hairpin loops of stem C decreased the ability of the tRNA-like structure to interact with RdRp. Furthermore, in the absence of the rest of the BMV tRNA, stem C is able to interact with RdRp. The addition of an accessible initiation sequence containing ACCA3' to stem C created an RNA capable of directing RNA synthesis. Synthesis from this minimal minus-strand template is dependent on sequences in the hairpin and bulged loops.     

The RNA replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis

Replication of the RNA genomes of tombusviruses, which are small plus-sense RNA viruses of plants, may be regulated by cis-acting elements, including promoters and replication enhancers that are present in the RNA templates. Using a partially purified RNA-dependent RNA polymerase (RdRp) preparation (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), we demonstrate that the minus-strand templates of tombusviruses contain a replication enhancer, which can upregulate RNA synthesis initiating from the minimal plus-strand initiation promoter by 10- to 20-fold in an in vitro assay. Dissection of the sequence of the replication enhancer element revealed that the two stem-loop structures present within the approximately 80-nucleotide-long enhancer region have interchangeable roles in upregulating RNA synthesis. The single-stranded sequence located between the two stem-loops also plays an important role in stimulation of RNA synthesis. We also demonstrate that one of the two hairpins, both of which are similar to the hairpin of the minus-strand initiation promoter, can function as a promoter in vitro in the presence of short cytidylate-containing initiation sites. Overall, the in vitro data presented are consistent with previous in vivo results (D. Ray and K. A. White, Virology 256:162-171, 1999) and they firmly establish the presence of a replication enhancer on the minus-stranded RNA of tombusviruses.